1. Field of the Invention
The invention in the field of biochemistry and medicine relates to anti-cancer compounds of the hydroxytolan family, and methods of killing tumor cells, inhibiting tumor growth and development, and treating cancer in subjects in need thereof using these compounds alone or in combination with ascorbate and/or and certain cyclic compounds.
2. Description of the Background Art
It is believed that 554,740 Americans died from cancer in 1996. Ten years later, the National Cancer Institute estimated that 570,280 Americans would die of cancer annually. Existing cancer treatment technologies clearly are not adequate. Despite notable progress, there continues to exist a need for better drugs and therapeutic modalities to combat cancer and improve the quality and the duration of life of cancer patients. Of particular interest are orally active drugs that possesses antitumor activity either alone or in conjunction with other chemotherapeutic or anticancer agents.
More than 1 million new cases of skin cancer were diagnosed in the United States in 2006 (Cancer Facts and Figures 2006, American Cancer Society). About 112,000 of these new cases were melanoma of which about 44% cases were noninvasive and about 56% were invasive. The latter were distributed as ˜55% in men and ˜45% women. Human melanoma, a potentially preventable malignancy, is the most serious skin cancer and is among the most drug resistant of all malignancies (Yang, S et al., 2005, Mol. Cancer. Ther. 4: 1923-35). Excluding basal cell and squamous cell carcinomas, which together are the most common cancers in both sexes, these figures make invasive melanoma the fifth most common cancer in men and the seventh most common cancer in women. Furthermore, 7,910 Americans (˜63% men and ˜37% women) died of melanoma in 2006, representing one death every 67 minutes). Additional figures indicate that the incidence of melanoma has increased 690% from 1950 to 2001, while the overall mortality rate increased only 165% during this same period (The Lewin Group, Inc., The Burden of Skin Diseases, 2004; Soc. for Invest. Dermatol. and Amer. Acad. Dermatol, pp. 1-110). After breast cancer in premenopausal women (ages 30-34 years), melanoma has exhibited the fastest rate of increase in incidence in the United States, while rates for many other cancers are falling (Demierre, M.-F et al. 2003, J. Clin. Oncol. 21: 158-65). The lack of efficacy of current treatment protocols points to the need for the development of effective modalities for treating or preventing melanoma.
Uncontrolled imbalance between cell proliferation and cell differentiation or cell death may result in the development of malignant or cancerous clones of cells which are commonly less differentiated than their normal counterparts. Thus, promising targets for cancer intervention are induction of (i) differentiation of pre-malignant or malignant cells into more normal cells and (ii) tumor-specific cell death during the process of carcinogenesis or tumor development. Compounds which induce differentiation or cell death are candidates for cancer chemoprevention and/or chemotherapy (Hong W K and Sporn M B, Science, 1997, 278:1073-7; Suh N et al., Anticancer Res., 1995, 15:233-9; Fimognari C et al., Biochem Pharmacol., 2004, 68:1133-8).
In the last several years, hundreds of plant extracts have been evaluated for their potential as cancer chemopreventive agents and for their ability to induce cell death (Clement M V et al., Blood, 1998, 92:996-1002; Cooke D et al., Eur J Canc., 2005, 41:1931-40). Because inflammation and reactive oxygen species (ROS) can be major determinants in the development of many diseases, including cancer, as well as in viral replication, plant polyphenols have been evaluated as chemopreventive agents because of their antioxidant, oxygen radical scavenging and anti-inflammatory activities.
Many of these compounds inhibit the cellular events associated with all 3 stages of carcinogenesis (initiation, promotion and progression). One strategy has employed phenolic compounds to counteract cancer formation by blocking one or several steps in this multistage process. A non-flavonoid polyphenol of the stilbene group, resveratrol (3,5,4′-trihydroxy-trans-stilbene, depicted below), is a typical example of such a compound. Resveratrol consists of two aromatic rings linked by an ethylene bridge with two hydroxyl groups at the 3 and 5 positions of one ring and one hydroxyl group at the 4′ position of the other ring.
Resveratrol occurs in a trans and a cis configuration, and as a glucoside. The trans isomer is most active while the cis-isomer is almost inactive in may types of biological and biochemical tests. Its diverse bioactivities include antioxidant action, modulation of lipid and lipoprotein metabolism, inhibition of platelet aggregation, vasorelaxing activity, anticancer activity and estrogenic activity (Aggarwal B B et al., Anticancer Res., 2004, 24:2783-40; Fremont L., Life Sci., 2000, 66:663-73). Resveratrol thus may act on cancer cells by promoting cell death, activating phase II detoxification and attenuating cell proliferation, DNA synthesis and inflammation (Aggarwal et al., supra; Aziz M H et al., Int J Oncol., 2003 23:17-28; Dong Z., Mutat Res., 2003, 523-524:145-50; Fremont L., supra; Gusman J et al., Carcinogenesis, 2001, 22:1111-7; Jang M et al., Science, 1997, 275:218-20; Savouret J F et al., Biomed Pharmacother., 2002, 56:84-7; Signorelli P et al., J Nutr Biochem., 2005, 16:449-66). The trans isomer of resveratrol blocks all 3 stages of carcinogenesis noted above.
Resveratrol was reported to inhibit cell proliferation and cause apoptotic cell death by modulating numerous mediators of cell cycle and survival signaling. Depending on concentrations, resveratrol “switched” cells between reversible cell cycle arrest and irreversible apoptosis. Specifically, resveratrol treatment blocked the cell cycle in the G0/G1, G1/S transition, S phase or G2/M phases by (a) suppressing cyclins and their corresponding kinases, (b) increasing p53 levels or (c) inhibiting DNA synthesis. Resveratrol also up-regulated pro-apoptotic members of the Bcl-2 family and down-regulated anti-apoptotic members of this family. Finally, resveratrol inhibits NF-κB and AP-1 signaling pathways, their upstream kinases and their downstream targets (including inducible cyclooxygenase-2, inducible nitric oxide synthase and matrix metalloprotease-9. Thus, resveratrol can inhibit proliferation and induce cell death.
Resveratrol is considered to be a phytoestrogen because of its structural homology to the estrogens, and its ability to compete with estrogens for binding to estrogen receptors and to activate receptor-mediated gene transcription. However, resveratrol also manifests anti-estrogen function and can inhibit hormone-induced carcinogenesis with agonistic or antagonistic hormonal activity depending on the intake concentration, tissue-specific expression of estrogen receptors, cofactors present for DNA binding and different gene promoters (Aggarwal et al., supra; Fremont, supra). Likewise, resveratrol represses transcription or translation of different classes of androgen up-regulated genes via a reduction in androgen receptor (AR) content (Mitchell S H et al., Canc Res, 1999, 59:5892-5).
While the antitumor mechanisms of resveratrol are pleiotropic, and it appears to be a promising antitumor agent in part because it affects the 3 stages of carcinogenesis, its use has been hampered by its relatively low aqueous solubility and its apparent lack of selectivity or specificity for tumor cells. Resveratrol is also significantly toxic to normal cells (Aggarwal et al., supra). The present invention is directed primarily to a distinct class of diphenol compounds, the tolans, and their advantageous properties as anti-cancer agents when compared to resveratrol.
A number of stilbenes that are tubulin-binding agents, e.g., combretastatin AI, combretastatin A4 (Chaplin, D J et al., Brit J. Canc 27, S86-88 (1996)) and combretastatin A4 phosphate (Chaplin, D J et al., Anticancer Res 19(1A), 189-96, (1999)) selectively damage neovasculature of solid tumors in animal models. Other analogues of combretastatin A4 show activity in assays of cytotoxicity in vitro and in animal tumour models. See, for example Cushman, M et al., 1991, J. Med. Chem. 34:2579-88; Ohsumi, K. et al., 1998, J. Med. Chem. 41:3022-32; Hatanaka T et al., 1998, Bioorg Med Chem. Lett. 8:3371-4; Woods, J A et al. 1995, Brit. J. Canc 71:705-11). However, it is not apparent whether such compounds act through direct effects on tumor tissue or on by selective anti-vascular mechanisms. International Patent Pub. WO 01/12579 discloses a series of cis-stilbenes with vascular damaging activity, particularly targeting newly-formed vascular endothelium, especially that associated with solid tumors. Such compounds were said to be useful in the prophylaxis and treatment of cancer (sold tumors) and in other diseases associated with undesired neovascularization such as diabetic retinopathy, psoriasis, rheumatoid arthritis, macular degeneration and the formation of atherosclerotic plaque.
U.S. Pat. Nos. 6,599,945 and 7,094,809 (co-invented by the present inventor) disclose several hydroxytolan compounds and their use in inhibiting the formation of infectious herpes virus particles or for treating gonorrhea caused by Neisseria gonorrhoeae. However the potential utility of these, or any other hydroxytolans as anti-cancer agents was unknown until the making of the present invention. U.S. Pat. Nos. 6,197,834 and 6,355,692 disclose certain hydroxylated stilbenes, and specifically resveratrol, for similar uses. The use of resveratrol in suppressing or treating cancer is also disclosed in U.S. Pat. No. 6,008,260. None of these references directed to resveratrol disclose the hydroxytolans of the present invention nor suggest the notion of their use as anti-cancer agents.
Vitamin C/Ascorbate
The chemical structure of Vitamin C (sodium ascorbate) is shown below:
Vitamin C (abbreviated herein as “VitC or “VC”) acts as a pro-oxidant, and has been evaluated as an antitumor agent. Several in vitro studies demonstrated that VC selectively accumulated in, and was toxic to, a variety of human tumor cells in culture. These included malignant melanoma cells, leukemia cells, neuroblastoma cells, ascites tumor cells as well as acute lymphoblastic leukemia, epidermoid carcinoma and fibrosarcoma cells.
Several case reports describe favorable outcomes in cancer patients undergoing high dose intravenous VC therapy. E. Cameron and L. Pauling (Proc Natl Acad Sci USA 73:3685-9, 1976) reported the effect of administering supplemental ascorbate (10 g/day intravenously (i.v.) for 10 days followed by 10 g/day orally thereafter) to 100 terminal cancer patients as part of routine management of these patients. The “controls,” 1000 subjects matched for age and sex, were left untreated. These were individuals who suffered from cancer of the same primary organ type and histological type as the patient group. The mean survival time (MST) for ascorbate-treated subjects (>210 days) was more than 4.2 times greater than that of the controls (50 days) (p<<0.0001). Six of the 100 treated subjects had ovarian cancer. When their progress was compared to that of disease-matched controls, the MST of 148 days was twice as long as the controls (MST: 71 days; p<0.005). The results suggested that VC may be of value in the treatment of advanced ovarian cancer. Two later randomized, double-blind, placebo-controlled, clinical trials (Creagen et al., New Eng. J. Med. 301:687-690, 1979; Moertel et al., New Eng. J. Med. 312:137-141, 1985) that were designed to evaluate the effectiveness of 10 g of oral VC in patients with advanced cancer, reported no benefits of oral VC treatment. More recently, these studies have been criticized (Riordan et al., Med. Hypotheses 44:207-213, 1995) because the oral VC dose of 10 g/day is not believed to be sufficient to achieve plasma concentrations that were found to be cytotoxic for tumor cells in culture. Finally, a number of case studies (Riordan et al., supra; Riordan et al., P.R. Health Sci. J. 23:115-118, 2004; Drisko et al., J. Am. Coll. Nutr. 22:118-23, 2003) reported the effects of high i.v. doses of VC in patients with breast, colorectal, ovarian, pancreatic, renal cell carcinoma. VC doses ranged from 10 to 100 g given twice per week with the majority of doses being 60-70 g per infusion. The results of these case reports suggested that high i.v. doses of VC do not interfere with conventional anticancer therapy; are generally not toxic to cancer patients with normal renal function; and induce a small number of complete remissions. This high dose i.v. regimen of VC administration, while manifesting antitumor activity, is financially burdened an inconvenient as it requires additional doctor visits.
VC usage in humans is well-documented, and the vitamin is well tolerated in animals. Mice given daily VC doses of 6.5 g/Kg body weight for 6 weeks and 2 g/Kg for 2 years showed no abnormal rates of mortality, weight changes, blood chemistry, hematology, histology, or other pathologies (Klenner, F R, 1951, South Med J 113:101-7). This reference includes a table of therapeutic doses ranging from 35 g/day for a 220 pound man to 1.2 g/day in infants. Also indicated were maintenance doses of 60 mg/kg/day (i.e., about 2180 mg/day) and 75 mg/day for these respective groups. The only systemic toxicity noted at these doses has been diarrhea/gastrointestinal upset, in which case the doses are injected, bypassing these complications.
NF-κB (Nuclear Factor-Kappa B)
NF-κB is a protein complex transcription factor. NF-κB and its inhibitor IκB form an inactive complex in the cytoplasm. Activation occurs by phosphorylation, ubiquitination and degradation of IκB, which then releases active NF-κB into the nucleus where it regulates the expression of target genes leading to a variety of cellular effects. Constitutively active NF-κB activates expression of genes that keep cells proliferating and protect them from conditions that would otherwise induce death.
Dysregulation of NF-κB has been linked to cancer. In tumor cells, NF-κB is active either due to mutations in genes encoding the NF-κB transcription factors themselves or in genes that control NF-κB activity (such as IκB genes). Some tumor cells secrete factors that activate NF-κB. Blocking NF-κB can stop tumor cell proliferation, induce cell death or increase the cells' sensitivity to the action of antitumor agents. Thus, NF-κB is a subject of active research as a target for anti-cancer therapy. As noted, The NF-κB family members play multiple roles in the regulation of immune and inflammatory responses, developmental processes in addition to cancer.
U.S. Pat. No. 6,410,516 (Jun. 25, 2002) to Baltimore et al. discloses—constitutive and tissue-specific protein factors that bind to transcriptional regulatory elements of Immunoglobulin (Ig_genes (promoter and enhancer). NF-κB, the gene encoding NF-kB, IκB and the gene encoding IκB are useful for enhancing transcription of Ig genes. Recent work by the laboratories of Karin, Ben-Neriah and others has highlighted the importance of NF-κB in inflammation as well as cancer, and has underscored the value of therapies that regulate NF-κB activity (Pikarsky E, Ben-Neriah Y., Eur J Cancer. 2006 42:779-84. Häcker H, Karin M., Sci STKE. 2006 Oct. 17; Karin M., Nature. 2006, 441:431-6; Karin M., Mol. Carcinog. 2006 45:355-61; Luo J L et al., J Clin Immunol. 2005, 25:541-50; Luo J L et al., J Clin Invest. 2005 115:2625-32; Karin M, Greten F R., Nat Rev Immunol. 2005; 5:749-59; Greten F R, Karin M. Cancer Lett. 2004; 206:193-9; Karin M et al., Nat Rev Drug Discov. 2004, 3:17-26; Lin A, Karin M., Semin Cancer Biol. 2003, 13:107-14; Amit S, Ben-Neriah Y. Semin Cancer Biol. 2003, 13:15-28; Karin M et al., Nat Rev Cancer. 2002; 2:301-10; Karin M, Lin A. Nat. Immunol. 2002, 3:221-7).
The discovery that activation of NF-κB nuclear translocation can be separated from the elevation of oxidant stress provides a basis for developing strategies for NF-κB inhibition. NF-κB has been shown as the only biomarker that can predict a risk of progression or recurrence of prostate cancer. US Patent Pub. 20050026196 discloses NF-κB as a prognostic marker for prostate cancer and for predicting risk of progression or recurrence by measuring the proportion of NF-κB localized in the nuclei of a tumor sample compared to total NF-κB in the tumor sample. Drugs which inhibit NF-κB within tumor cells are believed to be potentially useful anti-cancer therapeutics.
As noted above, resveratrol is widely recognized to have anti-cancer activity that also inhibits NF-κB. The present inventor discovered new compounds that may be considered resveratrol analogs; their actions on NF-κB activity are disclosed herein.
None of the documents cited above disclose or suggest the specific pharmaceutical compositions, methods and uses of the compounds that are disclosed and claimed herein. To the extent that any specific disclosure in these publications or other publications may be considered to anticipate any generic aspect of the present invention, the disclosure of the present invention should be understood to include a proviso or provisos that exclude any such species that were previously disclosed. The aspects of the present invention which are not anticipated by the disclosure of said publications are also unobvious from the disclosure of these publications, due at least in part to the unexpectedly superior results disclosed or alleged herein. One advantage of the present invention is that the hydroxytolan or other polyphenolic described herein acting in concert with Vitamin C or other ascorbates, including salts, with or without a cyclic compound described below, provides an improved anti-cancer composition or improved method for combination anti-cancer therapy.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.